Recent work from this laboratory has focused on the characterization of RNA processing enzymes from the Archaea (formerly called the Archaebacteria). As expected from their unique evolutionary position, these cells contain a mosaic of molecular characteristics. The enzymes that participate in tRNA maturation are a good example of this diversity. We have observed that the tRNA intron endonuclease from Haloferax volcanii has a substrate recognition mechanism that is distinct from its eukaroytic counterpart. This enzyme does not require mature tRNA structure, rather it recognizes a specific structure, and possibly sequences, a the exon-intron boundaries. We have also found that the RNaseP RNA of this organism is similar in structure and sequence to the bacterial RNaseP RNA. However, this RNA does not appear to have catalytic activity in the absence of protein as do the bacterial RNAs. The specific aims of the proposed research include a determination of the precise recognition properties of the Archaea tRNA intron endonuclease using in vitro and in vivo assays developed in this laboratory. Methods are described for the purification of the Methanosarcina barkeri tRNA intron endonuclease protein, and the cloning of its gene. The endonuclease purification studies are being done in collaboration with Dr. R. Garrett at the University of Copenhagen. Studies on the RNaseP RNA are also planned. We propose to reinvestigate the catalytic capability of the halobacterial RNaseF RNA using the small H. trapanicum RNaseP RNA, and to isolate the gene encoding the M. barkeri RNaseP RNA. These RNaseP RNAs will also be used to develop methods for the reconstitution of their holoenzyme complexes. These experiments will provide a valuable perspective on the evolution of RNA processing enzymes, and further our understanding of the factors that govern RNA protein interactions.